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UNITED STATES DEPARTMENT OF AGRICULTURE 
BULLETIN No. 844 

Contribution from the Bureau of Plant Industry 
WM. A. TAYLOR, Cliief 



Washington, D. C. 



PROFESSIONAL PAPER 



August 11, 1920 



SWEET-CLOVER SEED 

Part I. — Pollination Studies of Seed Production 
Part II. — Structure and Chemical Nature of the Seed Coat and 
its Relation to Impermeable Seeds of Sweet Clover 

By 

H. S. COE, formerly Assistant Agronomist, Office of Forage-Crop 
Investigations, and J. N. MARTIN, Professor of Mor- 
phology and Cytology, Iowa State College 



CONTENTS 



Page 

Part I.— Pollination Studies of Seed Pro- 
duction. 

Unsatisfactory Yields of Sweet-Clover 

Seed 1 

Previous Investigations of the Polli- 
nation of Sweet Clover .... 2 
Outlineof Pollinating Experiments . 8 
Structure of the Flowers of MelUotus 

alba 1 

Development of the Floral Organs of 

Sweet Clover 6 

Fertilization in Melilotus alba . . 8 
Development of the Seed .... 8 
Mature Pollen of Sweet Clover . . 9 
Germination of the Pollen .... 9 
Cross-Pollination and Self-Pollina- 

tion of Sweet Clover J.0 

Artificial Manipulation of Sweet- 
Clover Flowers 10 

Seed Production of Melilotus alba 

under Ordinary Field Conditions . 13 
Efficiency of Certain Kinds of Insects 
as Pollinators of Sweet Clover . . 14 



Page 



Part I.— Pollination Studies of Seed 
Production— Continued. 

Relation of the Position of the 
Flowers on Melilotus alba Plants 

to Seed Production 19 

Influence of the Weather at Blossom- 
ing Time upon Seed Production . ZO 
Insect Pollinators of Sweet Clover . 21 
Effect of Moisture upon the Pro- 
duction of Melilotus alba Seed . 23 
Part n. — Sirncture and Chemical Na- 
ture of the Seed Coat and Its Relation 
to Impermeable Seeds of Sweet Clover. 

Historical Summary 26 

Material and Methods 30 

Structure of the Seed Coat .... 31 
Microchemistry of the Seed Coat . 33 
The Seed Coat in Relation to the 

Absorption of Water 34 

A Comparison of Permeable and 

Impermeable Seed Coats ... 34 
The Action of Sulphuric Acid on the 
Coats of Impermeable Seeds . . 35 
Literature Cited 36 




WASmNGTON 

GOVERNMENT PRINTING OFFICE 

1920 



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1920 






UNITED STATES DEPARTMENT OF AGRICULTURE 




^•^/'^^u 



BULLETIN No. 844 

Contribution from the Bureau of Plant Industry 
WM. A. TAYLOR, Chief . 



Washington, D. C. 



PROFESSIONAL PAPER 




sJ^'^^^u 



August 11, 1920 



SWEET-CLOVER SEED. 

Part I. — Pollination Studies of Seed Production. 

Part n. — Structure and Chemical Nature of the Seed Coat and Its 
Relation to Impermeable Seeds of Sweet Clover. 

By H. S. Qo^, formerly Assistant Agronomist, Office of Forage-Crop Investigations, and 
J. N. Martin, Professor of Morphology and Cytology, Iowa State College, 



CONTENTS. 



Part I.— Pollination Stitdies of Seed 
Production. 
Unsatisfactory yields of sweet-clover seed . 1 
Previous investigations of the pollination 

of sweet clover 2 

Outline of pollinating experiments 3 

Structure of the flowers of Melilotus alba. . 4 
Development of the floral organs of sweet 

clover 5 

Fertilization in Melilotus alba 8 

Development of the seed 8 

Mature pollen of sweet clover 9 

Germination of the pollen. 9 

Cross-pollination and self-pollination of 

sweet clover 10 

Artificial manipulation of sweet-clover 

flowers 10 

Seed production of Melilotus alba under 

ordinary field conditions 13 

Efficiency of certain kinds of insects as 

pollinators of sweet clover 14 

Relation of the position of the flowers on 

Melilotus alba plants to seed production. 19 



Page. 
Part I.— Pollination Studies of Seed 
Production— Continued. • 
Influence of the weather at blossoming 20 

time upon seed production 21 

Insect pollinators of sweet clover 22 

Effect of moistiu-e upon the production of 

Melilotus alba seed 

Part II.— Structure and Chemical Na- 
ture OF THE Seed Coat and its Rela- 
tion to Impermeable Seeds of S-wiiet 
Clover. 

Historical summary 26 

Material and methods 30 

Structure of the seed coat 31 

Microchemistry of the seed coat 33 

The seed coat in relation to the absorption 

of water 34 

A comparison of permeable and imper- 
meable seed coats 34 

The action of sulphuric acid on the coats 

of impermeable seeds 35 

Literature Cited 36 



Part I.— POLLINATION STUDIES OF SEED PRODUCTION. 

UNSATISFACTORY YIELDS OF SWEET-CLOVER SEED. 

In some sections of the country much trouble has been experienced 
for a few years past in obtaining satisfactory yields of sweet-clover 
seed. This difficulty has been due for the most part to the following 
causes: (1) To cutting the plants at an improper stage of develop- 

153321°— 20— Bull. 844 1 



2 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

merit, (2) to the use of machinery not adapted to the handlmg of the 
crop, (3) to the shedding of immature pods, and (4) possibly to the 
lack of pollination. As the first two have been overcome, mainly 
because of a better understanding of the requirements for handling 
this crop, the subject matter of this bulletin is concerned primarily 
with the factors which produce the third and fourth causes. 

Where the production of seed was disappointing although the 
plants produced an abundance of flowers, it has been observed 
that many apparently were not fertilized, or if fertilized the pods 
aborted. In order to obtain data in regard to the causes of the 
failure of sweet clover to produce a normal seed yield, a study was 
made of the insects which were most active in pollinating the flowers, 
the source of the pollen necessary to effect fertilization, and the 
conditions under which the flowers must be pollinated in order to 
become fertilized. The relation of environmental conditions to the 
shedding of immature pods was also investigated. In order to 
overcome local environmental factors as much as possible, the 
experiments were conducted on the Government Experiment Farm 
at Arlington, Va., and in cooperation with the botanical department 
of the Iowa State College at Ames, Iowa. 

PREVIOUS INVESTIGATIONS OF THE POLLINATION OF SWEET CLOVER. 

Since Darwin (4, p. 360)^ published the statement that a plant of 
Melilotus officinalis protected from insect visitation produced but a 
very few seeds, while an unprotected plant produced many, other 
scientists have investigated this subject. Knuth (19, v. 1, p. 37), in 
giving a list of the best known cases of self-sterility in plants, men- 
tions Melilotus officinalis. The same author (19, v. 2, p. 282) states 
that since the stigma projects beyond the anthers, automatic self- 
pollination is difficult, and for the same reasons Miiller (29, p. 180) 
believes that self-fertilization is not apt to occur. 

In 1901 Kirchner (18, p. 7) covered a number of Melilotus alba 
racemes with nets. On one of the plants 12 protected racemes 
produced 187 seeds and on another plant only one seed was obtained 
from 10 covered racemes. This experiment was duplicated in 1904, 
with the result that 40 netted racemes produced an average of 38 seeds 
each. Kirchner concluded from this experiment that spontaneous 
seK-pollination occurs regularly even though the stigma projects 
above the anthers. He (18, p. 8) also performed an experiment with 
Melilotus officinalis in 1901. At this time 16 isolated racemes pro- 
duced a total of 11 seeds. This experiment was repeated in 1904, 
with the result that 16 protected racemes produced an average of 14 
seeds each. As the racemes on one of the plants that was protected 

' The serial numbers in parentheses refer to "Literature cited," pages 36-38. 



SWEET-CLOVER SEED. 6 

in 1904 died, Kirchner concluded that the flowers of M. officinalis 
were especially sensitive to inclosure in nets and that the failures to 
obtain more, than a very few seeds on protected racemes in Darwin's 
experiment and in his first experiment were due to this cause. 

According to Kerner (17, v. 2, p. 399) the peas and lentils (Pisum 
and Ervum) and the different species of horned clover and stone 
clover (Lotus and Melilotus) as well as the numerous species of the 
genus Trifolium and also many others produce seeds when insects 
are excluded from the plants, and only isolated species of these 
genera gave poor yields without insect visitation. 

OUTLINE OF POLLINATING EXPERIMENTS. 

The yield of sweet-clover seed varies greatly from year to year in 
many parts of the United States. It has been assumed that this 
variation was due to climatic conditions, -as excellent seed crops were 
seldom harvested in seasons of excessive rainfall or of prolonged 
drought just preceding or during the flowering period. The lack of 
a sufficient number of suitable pollinating insects also was thought 
to be an important factor in reducing seed production. This was 
especially true where the acreage of sweet clover was large and where 
few, if ahy, honeybees were kept. 

In order to obtain data upon the factors influencing the yield of 
seed, a series of experiments was outlined to determine (1) whether 
the flowers are able to set seed without the assistance of outside agen- 
cies, (2) whether cross-pollination is necessary, (3) the different kinds 
of insects which are active agents in pollinatuig sweet clover, and (4) 
whether a relation exists between the quantity of moisture in the soil 
and the production of seed. 

The racemes containing the flowers which were to be pollinated by 
hand were covered with tarlatan before any of the flowers opened and 
were kept covered except while being pollinated until the seeds were 
nearly mature. This cloth has about twice as many meshes to the 
linear inch as ordinary mosquito netting and served to exclude all 
insects that are able to pollinate the flowers. When entire plants 
were to be protected from all outside agencies, cages covered with 
cheesecloth, glass frames, or wire nettmg were used. 

A preliminary study of the pollination of Melilotus alba and M. 
officinalis showed that both were visited by the same kinds of insects 
and that both required the same methods of pollination in order to 
set seed. On this account M. alba was used in most of the experi- 
ments reported in this bulletin. Where M. officinxilis was employed 
it is so statedo 



4 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

STRUCTURE OF THE FLOWERS OF MELILOTUS ALBA. 

The racemes of Melilotus alba contain from 10 to 120 flowers with 
an average of approximately 50 per raceme for all of th^e racemes of 
a plant growing under cultivation in a field containing a good stand. 




Fig. 1.— Diflerent parts of the flower of Melilotus alba: 1, Side view of the flower; 2, side view of the flower 
with the carina and alae slightly depressed; 3, side view of the flower, showing the carina and alse de- 
pressed sufflciently to expose the staminal tube and the tenth free stamen; 4, ala; 5, alae and carina 
spread apart to show their relative position and shape; fi, flower after the petals have been removed, 
showing in detail the calyx and staminal tube; 7, the staminal tube split open to show the relative size 
and position of the pistil, a, Alse; b, vexillum; c, carina; d, calyx; c, stigma; 6, anthers; g, tenth free 
stamen; h, digitate process of the superior basal angle of an ala; i, depressions! in the ala; j, staminal 
tube; k, pistil. 

The flower consists of a green, smooth, or slightly pubescent calyx 
with 5-pointed lobes and with an irregular white corolla of five petals. 
(Fig. 1 .) The claws of the petals are not united nor are they attached 
to the staminal tube which is formed by the union of the filaments of 
the nine inferior stamens. As the claws of the alee and carina are not 



SWEET-CLOVER SEED. 



attached to the staminal tube, the petals may be bent downward 
sufficiently far so that many different kinds of insects may secure 
without difficulty the nectar secreted around the base of the ovary. 

The fingerlike processes of the alee are appressed closely to the 
carina, therefore the alae are bent downward with the carina by 
insects. These processes grasp the staminal tube superiorly and 
tightly when the carina and alse are in their natural positions, but 
when the carina is pressed downward by insects the fingerlike proc- 
esses open slightly but not so far that they do not spring back to their 
original position when the pressure is 
removed. The staminal tube splits 
superiorly to admit the tenth free 
stamen. The filament of this superior 
stamen lies along the side of this 
staminal tube. The filaments of the 
nine stamens which compose the stami- 
nal tube separate in the hollow of the 
carina. All stamens bear fertile an- 
thers. The pistil is in the staminal 
tube, the upper part of the style and 
stigma of which is inclosed with the 
anthers in the carina. The stigma 
is slightly above the stamens. 

An insect inserts its head into a 
sweet-clover flower between the vexil- 

lum and carina, the stigma, therefore, Fig. 2.— Lengthwise sectional view of a very 

comes into direct contact with the 
head of the insect and cross-pollination 
is effected. At the same time the an- 
thers brush against the insect, so that 
its head is dusted with pollen, to be 
carried to other flowers. 




young flower of Melilotus oZ6a, showing the 
relative development of the stamens and 
pistil. In the upper set of stamens the di- 
visions of the mother cells are completed, 
while division is just beginning in the 
lower set of stamens. In the ovules the 
outer integuments are well started on their 
development, a, Anther; o, ovule; p., 
pistil. X38. 



DEVELOPMENT OF THE FLORAL ORGANS OF SWEET CLOVER. 

The stamens of Melilotus alba and M. officinalis may be divided 
into two sets, according to their length and time of development. 
(Fig. 2.) The longer set extends about the length of the anthers 
above the shorter set, and the pollen mother ceUs in the longer set 
divide to form pollen grains at least two days earlier than those in 
the shorter set. At the time the pollen mother cells divide, the 
longer set of stamens is approximately three-eighths of a millimeter 
in length and the pistil about half a milUmeter long. The stigma 
and a portion of the style project beyond the stamens, and this rela- 
tive position is maintained to maturity. The pollen mother cells 
undergo the reduction division while the megaspore mother cells are 



6 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

just being clifTerentiated and wliile the outer integuments are barely 
prominent at the base of the nucellus. The pollen grains are formed 
while the embryo sac is beginning to develop. The division of the 
megaspore mother cell does not occur until a number of days later, 
and the embryo sac is not mature until the flower is nearly ready to 
open. Thus, the ])ollen grains are formed a week to 10 days before 
the embryo sac is ready for fertilization. The pollen grains increase 
in size and undergo internal changes after their formation. These 
changes, which are not completed until the flower is one-half or more 
of its mature length, may be regarded as the ripening processes, and 
they are undoubtedly necessar}'^ before the pollen is capable of func- 
tioning. For this reason it is probable that the pollen grains are not 
able to function much before the embryo sac is mature. 

The pistils of Meli- 
lotus alha and M. offic- 
inalis are straight for 
the greater part of 
their length, but curve 
rather abruptly toward 
the keel just below the 
capitate stigma. The 
surface of the stigma 
is papillate. (Fig. 3.) 
In their reaction with 

Fig. 3.-Stigma at the time of pollination, showing its papUlate ^udan 111, alkaum, and 
character and the position of the pollen in reference to the papillae safrauiu the Walls of 
inpollmation. X175. the papillae of the stig- 

ma show that some fatlike substances are present. Aside from water, 
the contents of the papillre consist chiefly of a fine emulsion of oil. 

DEVELOPMENT OF THE OVULES. 

The number of ovules in the ovary of Melilotus alba varies from 
two to five; however, most commonly, three or four ovules occur. 
In Melilotus officinalis the number in each ovary ranges from three 
to six. In both species the ovules are campylotropous at maturity 
with the micropylar end turned toward the base of the ovary. 

"Mature ovules contain two integuments, but the inner one does 
not close entirely around the end of the nucellus. The outer integ- 
ument develops considerably ahead of the inner one. The outer 
integument is much thickened at the micropylar end, the seed coat 
is formed from it, and the inner integument is used as nourishment 
by the endosperm and embryo. 

The number of megaspore mother cells in an ovule varies from 
one to many. Two or more embryo sacs often start to develop in 
the same ovule, but seldom more than one matures. (PI. I, figs. 1, 




SWEET-CLOVER SEED. 



2, and 3.) In general, the development of the embryo sac proceeds 
in the ordinary way, as described by Young (44, p. 133), with the 
inner megaspore functioning. (Text fig. 4 and PI. II, fig. 1.) In its 
development the nucellus is destroyed rapidly, the destruction being 
most rapid first at the micropylar end proceeding backward. The 
nucellus is completely destroyed at the micropylar end b}^ the time 
the embryo sac is mature, and consequently the embryo sac comes in 
contact with the outer integument in this region. (PI. II, fig. 1.) 
As the destruction of the nucellus extends toward the chalazal end 
the embryo sac becomes much elongated and tubelike. The antip- 
odals disappear early, so that a mature embryo sac consists of the 
egg, the synergids, and the two polars. The two polars lie in contact 
in the micropylar end of 
the sac near the egg until 
fertiUzation. 

STERILITY OF THE OVULES. 

In Melilotus alha and M. 
officinalis there is very 
Httle tendency toward 
sterility of ovules. In an 
extended study of ovules 
developing under normal 
and under excessive mois- 
ture conditions only an 
occasional one was found 
in which no reproductive 
cells were differentiated, 
and no ovaries were found 
in which all of the ovules 
were sterile. 




Fig. 4.— Median section through an ov'iile, showing the embryo 
sac with four nuclei and the position of the integuments. 
X150. 



DEVELOPMENT OF THE POLLEN. 



The pollen mother cells do not separate, but previous to the reduc- 
tion division the protoplasm shrinks from the walls, thus forming a 
dense globular mass which often occupies less than half the lumen of 
the mother cell. (PI. I, fig. 4.) Nuclear division occurs while they 
are in this contracted condition, and four nuclei are formed from two 
successive divisions. The cytoplasm is equall}^ distributed around 
each nucleus. The four masses of protoplasm separate, and as they 
enlarge a number of times and develop into mature pollen grains they 
become binucleate, and a wall is gradually formed around each. 
(PL I, figs. 5 and 6.) At first the cytoplasm is quite dense and con- 
tains some starch but no fatty oils. However, the cytoplasm of 



8 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE, 

mature pollen grains is vacuolate, and it contains a fatty oil in the 
form of an emulsion. Soon after the pollen grains are formed, the 
walls of the mother cells disappear, thus permitting the pollen grains 
to lie loose in the anther. 

FERTILIZATION IN MELILOTUS ALBA. 

The time intervening between pollination and fertilization was 
investigated with both self -pollinated and cross-pollinated flowers. 
In cross-pollination the parents were separate plants. This point 
was investigated with plants out of doors during the summer of 1916 
and with plants in the greenhouse during the following winter. The 
time elapsing between pollination and fertilization ranged from 50 to 
55 hours and was not longer in the case of self-pollinated than with 
cross-pollinated flowers. Furthermore, the rate of the development 
of the embryo in each kind of pollination was studied and was found 
to be as rapid in self-pollination as in cross-pollination. Therefore, 
self-pollination is apparently as effective as cross-pollination in 
Melilotus alba so far as the vigor of pollen tubes and the rate at which 
embryos develop are concerned. Melilotus offi^cinalis was not studied 
in reference to this point. 

Considerable difference often exists in the size of the young embryos 
in the ovides of the same pod. This is due in part to a difference 
in the time of fertilization, although some of it is due to a difference 
in noui'ishment. It was observed that the ovule first fertilized 
might be an upper one, lower one, or any one between these. Occa- 
sionally one or more ovules are not fertilized. 

DEVELOPMENT OF THE SEED. 

A proembryo with a rather long suspensor is developed from the 
fertilized egg. (PI. II, fig. 2.) The endosperm, which quite early 
forms a peripheral layer around the entire embryo sac, develops most 
rapidly about the embryo, which soon becomes thoroughly embedded 
in it. (PI. Ill, figs. 1 and 2.) After the embryo has used up the 
endosperm in the micropylar end and has enlarged so much as to 
occupy nearly all of the space in this region, the development of the 
endosperm becomes more active in the chalazal end, and when the 
embryo is mature there is very little endosperm left. 

The seed coat begins to form about the time of fertilization, 
although it apparently does not depend upon it, for in ovules where 
fertilization is prevented the outer integument undergoes the early 
modifications in the development of the seed coat before the ovule 
breaks down. The development of the seed coat is apparent first at 
the micropylar and chalazal ends, where the outer cells of the outer 
integument become much elongated and their outer walls thicken 
very soon after fertilization. The modifications in the development 



Bui. 844, U. S. Dept. of Agriculture. 



Plate I. 





Development of the Ovules and Pollen in Sweet Clover. 

Fig. 1.— Section through the nucellus of an ovule of Melilotus alba, showing two megaspore mother cells. 
X360. Fig. 2.— Median section through an ovule of Melilotus alba, showing the two cells resulting 
from the first division of the megaspore mother cell, and the relative development of the different 
parts of the o^■ule. X300. Fig. 3.— Section through the nucellus of an ovule of Mdilotus alba . show- 
ing two embryo sacs, one being more advanced than the other. X360. Fig. 4. — Protoplasm of the 
pollen mother cell of Melilotus alba contracted and ready to undergo division. X560. Fig. 5. — Pollen 
grains of Melilotus alba just formed, showing their dense cvtoplasm and the presence of the mother- 
cell wall. X560. Fig. 6.— o, Mature pollen grain of Melilotus alba, showing the binucleate condition 
at the time of shedding; b, surface view. X560. 



Bui. 844, U. S. Dept. of Agriculture. 



Plate II. 




Fig. I. — Median Section through an Ovule of Melilotus alba. 

The embryo sac is shown ready for fertilization. The egg and synergids are in contact with the 
outer integument at the micropylar end. The remains of the" antipodals may be seen at the 
chalazalend. X180. 




Fig. 2. — Section through an Ovule of Melilotus alba, about Three 
Days After Fertilization. 

The proembrvo, the endosperm, and modifications of the integuments are shown. At this stage 
the suspens'or is a prominent part of the proembryo, and the endosperm is most abundant around 
the embryo. The inner integument is being rapidly destroyed, and the outer integument is 
beginning to form the seed coat, as is indicated by the modifications in the outer layer of its cells, 
which are elongating and thickening their outer walls. X33. 



Bui. 844, U. S. Dept. of Agriculture. 



Plate III. 




Fig. I. — Section of an Ovule of Melilotus alba after Fertilization. 

The stage of development is a little later than that shown in Plate II, figure 2. The embryo is 
embedded deeply in endosperm tissue. X45. 




Fig. 2. — Section through an Ovule of Melilotus alba after the Embryo 
IS Nearly Half Mature. 

But little endosperm remains except in the chalazal end, and very little remains of either the 
nucellus or inner integument. The modifications which transform the outer integument into a 
seed coat are well upder way. Not only the outer layer of cells which becomes the Malpighian 
layer is quite well modified, but also the layer beneath is being transformed into the osteosclerid 
layer. X30. 



Bui. 844-, U. S. Dept. of Agriculture. 



PLATE IV. 




Stubble of Melilotus alba. 

These plants , which were cut 12 inches above the ground during rainy weather, had made a 40 to 42 inch 
growth. The stubble became infected at the top and the light-colored portions of them were killed by 
disease, thus checking the water supply to the growing branches above the infection. 



SWEET-CLOVER SEED. 



9 



of the seed coat extend around the ovule from these points, involving 
at first only the outer or epidermal layer of cells which form the 
malpighian layer. Later, the cells just beneath the malpighian layer 
form the osteosclerid layer. Accompanying or closely folkm-ing the 
formation of the osteosclerid cells, the remaining cell layers of the 
outer integument become modified into the nutritive and aleurone 
layer, and the seed coat is fully formed. ^leantime the inner integu- 
ment is practically all used as food. 

MATURE POLLEN OF SWEET CLOVER. 

The pollen grains of Melilotus alha and of M. officinalis are quite 
similar. Each grain contains three germ pores, and when viewed 
so that the pores are visible they present a slightly angled appearance. 
The average dimensions of the pollen of Melilotus alha and of J/, offici- 
nalis are 26 by 32 microns and 24 by 30 microns, respectively, when 
measured in the positions shown in h in Plate I, figure 6. 

The walls of the pollen grains have cutin deposited in them, as 
shown by their reactions with Sudan III, alkanin, safranin, and 
chloriodid of zinc. The contents of the pollen gi-ains give a distinct 
reaction when tested for fat, and Millons reagent shows that also 
some protein is present. Tests for sugars and starch showed that 
these substances are not present in perceptible quantities in mature 
pollen grains, although some starch is present in immature pollen. 

GERMINATION OF THE POLLEN. 

The germination of the pollen of Melilotus albei permits considerable 
variation in moisture, as is illustrated in Table I. 

Table I. — Germination of the -pollen of Melilotus alba in water and in solutions of 
cane sugar of different strengths . 



Melilotus alba. 


Pure 
water. 


Cane sugar in solution (per cent). 


8 


12 


18 


21 


30 


35 


4.5 


55 


Germination of pollen per cent . . 33 


23 


64 


4G 


60 


40 


31 


22 






The results given in Table I represent the average of 12 tests. 
Some of the pollen grains burst in pure water and in the weak cane 
sugar solutions, the percentage of bursting being greatest in pure 
water and decreasing as the percentage of sugar in the solution was 
increased. There was considerable variation in the percentages of 
germination in both water and in the solutions of different strengths, 
and at times there was very little bursting which was not accompanied 
by a high percentage of germination. The pollen tubes gi-ew as 
rapidly in water as in any of the sugar solutions, some reaching a 
153321°— 20— BuU. 844 2 



10 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

length of 100 microns in six hours. As the pollen tubes made no 
more growth in the solutions of sugar than in water, it is evident 
that the sugar is not used as food, but helps in germination by reducing 
the rate at which water is absorbed. 

To judge from Table I, the pollen of sweet clover can be effective 
not only under ordinary conditions but also when the flowers are 
wet with rain or dew or when the stigma is so dry that in order to 
obtain water from the papillae the pollen must overcome a high resist- 
ance offered by the sap of the papillae, a resistance that may be 
equal to the osmotic pressure of a 45 per cent solution of cane sugar. 
This is in accord with results obtained under field conditions, as 
flowers that were pollinated while rain was falling set seed satisfac- 
torily, indicating that a high percentage of humidity in the atmosphere 
does not check the germination of the pollen sufficiently to interfere 
with fertilization. Neither was the setting of seed affected when the 
soil about the roots of plants was kept saturated with water, showing 
that the excessive quantity of water in the stigmas resulting from an 
abundance of water in the soil did not interfere with the fertilization 
of the flowers. 

No definite counts were made of the germination of the pollen of 
Melilotus officinalis in the solutions of cane sugar of different strengths, 
but observations show that the moisture requirement of the pollen 
of this species is approximately the same as that of Melilotus alba. 

CROSS-POLLINATION AND SELF-POLLINATION OF SWEET CLOVER. 

Results published by previous investigators on the cross-pollina- 
tion and self-pollination of sweet clover do not agree. The experi- 
ments of Darwin (4) show that the flowers are self-pollinated to 
only a small extent. On the other hand, Kirchner (18) and Kerner 
(17) find that self-pollination occurs generally and that cross-polli- 
nation is not necessary for the production of seed. However, all 
investigators agree that many different kinds of insects are able to 
pollinate sweet clover. 

Because of the diverse opinions as to the pollination of sweet clover, 
a number of experiments were conducted to determine (1) whether 
insect visitation was necessary to pollinate the flowers, (2) if neces- 
sary, whether the flowers must be cross-pollinated, and (3) what 
insects are active agents as pollinators of sweet clover. 

ARTIFICIAL MANIPULATION OF SWEET-CLOVER FLOWERS.^ 

Experiments were conducted to determine, if possible, the effect 
of various types of artificial manipulation of sweet-clover flowers 
when in full bloom on the production of seed. Only healthy, vigor- 

iThe writers wish to acknowledge their indebtedness to Mr. Carl Kurtzweil for assistance in conducting 
part of the field experiments at Ames. 



SWEET-CLOVER SEED. 



11 



ous plants growing on well-drained soil were selected for these ex- 
periments. Before any of the flowers were open, the individual 
racemes were covered with tarlatan and labeled. (Fig. 5.) As soon 
as part of the flowers opened, the racemies were uncovered and after 
removing all flowers that were not open the open flowers were polli- 
nated and the racemes re-covered. If the flowers of sweet clover 
are not fertilized they will remain open for two to three days, then 
wither, and in a short time drop. But after being fertilized the ovules 




Fig. 5.— Individual racemes of white sweet clover covered with cheesecloth to protect them from insect 

visitation. 

enlarge very rapidly, and the corollas usually drop in about seven 
or eight days. Therefore, all fertilized flowers can be distinguished 
a few days after fertilization has taken place. Counts were made of 
the number of pods which formed in 10 to 12 days after poUination. 
An outline of the experiments is given in Table II. 

Table II. — -Treatment cf siveet-clover flowers in the artificial-manipulation experiments. 



Experiment. 



Method of pollin;^ting the flowers. 



A 
B 
C 

D 
E 

F. 



Check— covered. 

Check— open to insect visitation at all times. 

A separate toothpick was used to spring the keel of each flower on the raceme. 

One toothpick was used to spring the keels of ail the flowers on a raceme. 

Cross-poUinated. 

Raceme rolled several times between thumb and linger. 



12 BULLETIN- 844, U. S. DEPARTMENT OF AGRICULTUEE. 

As insects, and especially honeybees, usually visit all recently 
opened flowers on a raceme, experiments C and D were conducted to 
determine whether more seed would be produced when pollen from 
other flowers on the same raceme was placed on the stigmas of the 
flowers than when only the pollen produced by each flower was placed 
on its own stigma. The effect of polhnation when only the pollen 
produced by an individual flower was placed on its own stigmas was 
also obtained in experiment F, as by this method of pollination no 
pollen was transferred from one flower to another. It can not be 
stated definitely that the seed produced by the cross-pollinated 
flowers was the result of fertilization with foreign pollen, as the 
anthers were not removed from the flowers pollinated because it 
would be necessary to remove the anthers when the flowers were not 
more than two-thirds mature, and in doing this the flowers would be 
so mutilated that only occasionally would pollination at this time 
or at a later date be effective. The flowers listed in experiment E 
were pollinated a short time before they opened, and in each case 
pollen taken from flowers of other plants was placed on the stigmas. 
The petals of the cross-pollinated flowers were not mutilated, and 
in each case they returned to their original positions soon after polli- 
nation. The results obtained in experiment B, where the racemes 
were simply labeled and left open to the action of insects at all times, 
serve for comparison with other experiments where the flowers were 
protected from insect visitation and were artificially manipulated. 

Martin (25) found the setting of alfalfa seed and Westgate (40) 
found the setting of red-clover seed to be affected by an excessive 
quantity of moisture in the soil or atmosphere. In order to over- 
come the possible effect of this or of other detrimental factors, in 
each experiment only the flowers on a certain number of racemes 
were pollinated at one time. All of the experiments were repeated a 
number of times during the months of July and August, 1916, and 
the results given in Table III show the total number of flowers polli- 
nated and the number of pods that formed during the two months. 

The results presented in Table III show that flowers fertilized 
with pollen transferred from another plant produced a higher per- 
centage of pods than any of the other treatments. The results ob- 
tained in experiment D, where the same toothpick was used to 
spring the keels of all the flowers on a raceme, show that this method 
of pollination produced an average of 7.24 pods per raceme more than 
the racemes in experiment C, where a separate toothpick was used 
for each flower. These results indicate that pollen transferred from 
one flower to another on the same raceme is more effective than when 
the pollen produced by an individual flower is used to fertilize its 
own stigma. However, the results of experiment C prove that self- 
pollination is effective in Melilotus alba. In experiment B, which 



SWEET-CLOVER SEED. 



13 



was the open check, 4.3 per cent more flowers set seed than on the 
racemes where the same toothpick was used to spring all the keels, 
but 11.57 per cent more seed w^as obtained than in experiment C. 
Spontaneous self-pollination occurs to only a very small extent, as 
will be seen from the results of experiment A, in which an average of 
only 2.9 per cent of the flowers set seed. 

Table III. — Effect of different types of artificial manipulation on the seed production 
of sweet clover at Arlington, Va., and at Ames, Iowa, in 1916. 



Location. 



Experi- 
ment. 



Total number of - 



Racemes. 



Flowers. 



Pods set. 



Flowers that set 
seed (per cent). 



At each 
station. 



Average. 



Arlington 
Ames 

Arlington 
Ames 

Arlington 
Ajnes 

Arlington 
Ames 

Arlington 
Ames 

Arlington 



100 
196 



50 



3,510 
4,536 

5,599 
1,276 

1,229 
289 

1,480 
575 

377 
175 



144 
92 



3,973 
600 



701 
133 



936 
342 



307 
80 



4.1 
2.0 



70.95 
47.02 



57. 03 
46.02 



63.24 
59.47 



81. 43 
45.71 



2.9 
66.51 
54.94 
62.18 
70.10 



SEED PRODUCTION OF MELILOTUS ALBA UNDER ORDINARY FIELD 

CONDITIONS. 

The production of seed of Melilotus alba under ordinary field con- 
ditions varies considerably, not only in different parts of the country 
but also on different fields in the same region. A number of factors 
contribute to this variation, one of the most important of which 
appears to be the inability of the plant to supply all the developing 
seed with sufficient moisture, causing some of them to abort. As 
pointed out on page 22 this condition was very marked in certain 
parts of the country in 1916. However, poor seed production is 
not always correlated with lack of moisture, for the seed crop was a 
failure in 1915, where cloudy and rainy weather prevailed much of 
the time the plants were in bloom. It is believed that the lack of 
pollination by insects was the principal cause for the failure of seed to 
set, as very few insects visit sweet-clover flowers when such condi- 
tions prevail. As sweet-clover pollen will germinate in pure water 
and as plants which have their roots submerged in water set seed 
abundantly when pollinated, the failure of the seed crop in 1915 was 
not due to excessive moisture. 

As a rule, thin stands of sweet clover produce raore seed to the 
acre than thick stands and isolated plants more seed than those 
growing in either a thick or thin stand. The correlation of seed 



14 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

production with the thickness of stand is probably due to the shading 
and partial prevention of insect visitation to part of the racemes on 
the lower branches. Most of the flowers upon the lower branches 
of isolated plants are directly exposed to sunlight and to insect visits ; 
therefore the racemes on these branches produce as large a percent- 
age of seed as the racemes on the upper branches. In a thick 
stand, little seed is produced by racemes on the lower branches. 

A plant approximately 3 feet high growing close to the center of a 
field at Arlington, Va., in which was an average stand of four sweet- 
clover plants to the square foot was selected in order to determine 
the number of racemes produced and the average number of seeds 
to the raceme. This plant produced 196 racemes, which contained 
an average of 20.4 pods each. The racemes varied from 2 to 10 cm. 
in length, and the number of pods to the raceme ranged from to 75. 
The racemes on the upper and most exposed portions of the plants 
were larger and the flowers produced a much higher percentage of 
pods than the racemes close to the bases of the larger branches. 
Many of the small racemes on the lower branches produced less than 
five pods each. 

The data obtained from the two plants at Arlington that were 
protected from night-flying insects may also be cited here, as the 
results of that experiment show that night-flying insects are not an 
important factor in the production of sweet-clover seed, and, further, 
because they were growing imder the same conditions, in the same 
plat, and were approximately of the same size. These two plants 
produced a total of 544 racemes, with an average of 20.9 pods each. 
The number of pods to the raceme varied from to 86. 

EFFICIENCY OF CERTAIN KINDS OF INSECTS AS POLLINATORS OF SWEET 

CLOVER. 

In order further to test the self-sterility of sweet clover and to de- 
termine the relative efficiency of night-flying and of different 
kinds of day-flying insects as poUinators of the flowers, a number of 
cages covered with cheesecloth, glass, or wire screen having 14 
meshes to the linear inch were placed over plants at Arlington, Va., 
and at Ames, Iowa, in July and August, 1916. The bases of the 
cages were bm"ied several inches in the ground, so that insects could 
not pass under them. Cheesecloth was used to cover most of the 
cages and was made into sacks of such a size that they could be put 
on or removed from the frames of the cages without difficulty. It 
was stretched tightly over the frames and fastened to their bases 
with laths. 

A cage having two sides and the top of glass but with ends covered 
with cheesecloth to permit ventilation was used at Ames to protect 
a number of plants from insect visitation at all times. The purpose 



SWEET-CLOVER SEED. 15 

of this cage was to determine whether the partial shading of the 
plants in the cages covered with cheesecloth would have any effect 
upon the setting of seed. 

The cage covered with wire netting having 14 naeshes to the linear 
inch was used to determine the efficiency as pollinators of sweet 
clover of insects so small that they could pass through openings of 
this size. 

The plants used in the experiments at Arlington were growing 
close to the center of a field of sweet clover. Volunteer plants in a 
field that contained only a scattering stand were used at Ames. The 
cages were placed over the plants in all of these experiments before 
any of the flowers opened, and the work was continued until they 
were through blooming. 

PLANTS SUBJECT TO INSECT VISITATION AT ALL TIMES. 

A plant subject to insect visits at all times and growing in the same 
plat as those inclosed in the cages at Aiiington was selected as a 
check to those inclosed in the cages during their entire flowering 
period or for only a portion of it. This plant, which was in bloom at 
the same time as those inclosed in the cages, produced 196 racemes 
with an average of 20.4 pods each. As all of the racemes were col- 
lected and as those on the lower portions of the plant were smaller 
than those on the upper branches, the average number of seeds per 
raceme is much lower than it would have been if only the larger 
racemes had been collected. 

An isolated plant that was subject to insect visits at all times was 
selected for a check to the cage work conducted at Ames. This was 
necessary in order to get results that would be comparable with those 
obtained from the plants inclosed in the cages, as the cage experi- 
ments at Ames were conducted with isolated plants. The plant pro- 
duced 239 racemes, with an average of 41.6 pods. 

PLANTS PROTECTED FROM INSECT VISITATION DURING THEIR ENTIRE FLOWERING 

PERIOD. 

On July 3, 1916, a cage 3 feet square and 3 J feet high, covered with 
cheesecloth, was placed over three sweet-clover plants at Arlington. 
(Fig. 6.) This cage was not opened until August 3, when practically 
all of the racemes had passed the flowering stage and the few seeds 
that formed on some of them were practically mature. The three 
plants inclosed in the cage produced 904 racemes, with an average 
of 0.63 pod each. No pods were produced on 594 racemes, while 150 
produced but one each. None of the racemes produced more than 
five pods. 



16 



BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 



This experiment was duplicated at Ames in August, 1916, with the 
result that the three protected plants produced a total of 776 racemes, 
with an average of 0.19 pod each. 

The plants inclosed at Arlington produced 0.44 pod to the raceme 
more than the plants inclosed at Ames, and the average for the six 
plants at Arlington and at Ames is only 0.42 pod to the raceme. 
Results given below for nine plants inclosed in the glass-covered cage 

show that the pods 
produced per raceme 
by different plants 
varied from. 0.1 to 
0.45, which is slightly 
less than the varia- 
tion in the two cages 
covered with cheese- 
cloth. 

In order to deter- 
mine whether the 
shading of the plants 
in the cheesecloth- 
covered cages had 
caused the produc- 
tion of seed to be re- 
duced, a cage 4 feet 
wide, 4 feet high, and 
10 feet long, having 
glass sides and top, 
but with ends covered with cheesecloth to permit ventilation, was 
placed over nine plants at Ames in August, 1916. The results 
obtained in this experiment are presented in Table IV. 

Table IV. — Production of sweet-clover seed hy plants protected from insect visitation 
during their entire flowering period at Ames, Iowa, in 1916. 




Fig. 6.— Cage covered with cheesecloth to protect plants from insect 
visitation. 



Plant. 


Racemes 

per 

plant. 


Pods pro- 
duced 
by all 

racemes. 


Average 
number 
of pods 
to the 
raceme. 


Plant. 


Eacemes 

per 

plant. 


- 

Pods pro- 
duced 
by all 

racemes. 


Average 
number 
of pods 
to the 
raceme. 


1^0.1 


84 
130 
166 
199 
243 
131 


17 
58 
30 
88 
35 
36 


0.20 

.44 
.18 
.44 
.14 
.27 


No. 7 


119 

182 
340 


13 
83 
142 


0.10 


No. 2 


No. 8 


.45 


No.3 


No. 9 


.41 


No 4 


Total 




No.5 


1,594 


502 




No. 6 


.31 













The results given in Table IV show that an average of 0.31 of a 
pod to the raceme was obtained from 1,594 racemes and that the 
variation in seed production of the different plants was from 0.1 to 
0.45 to the raceme. The average seed production for the nine plants 



SWEET-CLOVER SEED. 17 

is 0.11 seed to the raceme less than the average results obtained from 
the six plants that were covered with cheesecloth. As this difference 
is well within the limit of variation for individual plants, it may be 
stated that the shading of the plants in the cheesecloth-covered cages 
did not reduce the production of seed. The results of this experiment 
show that spontaneous self-pollination does not occur regularly, as 
stated by Kirchner. 

FLOWERS POLLINATED ONLY BY NIGHT-FLYING INSECTS. 

In order to determine the importance of night-flying insects as 
pollinators, two cheesecloth-covered cages 3 feet square and 3 J feet 
high were placed over sweet-clover plants at Arlington on July 10, 
1916. The covers of the cages were removed each evening at 7:30 
and replaced each morning at 4:30 o'clock. Practically all the 
flowers on these plants had bloomed by August 2, and the seed pro- 
duced was nearly mature. The few racemes that contained opened 
flowers or buds were discarded. The three plants in one cage pro- 
duced 723 racemes, with an average of 3.76 pods each, while the one 
plant in the other cage produced 227 racemes, with an average of 
3.58 pods to the raceme. The four plants, therefore, produced a 
total of 950 racemes, with an average of 3.71 pods each. The only 
night-flying insect found working on sweet clover while these plants 
were in bloom was Diacrisia virginica Fabr. 

This experiment was duplicated at Ames in August, 1916, with the 
result that one plant subject to visitation only by night-flying insects 
produced 486 racemes, with an average of 16.5 pods each. 

The results obtained in these experiments show that night-flying 
insects were much more active in pollinating sweet clover at Ames 
than at Arlington. However, as the results obtained from the plants 
subject to visitation by day-flying insects only were practically the 
same as those obtained from plants which were subject to insect 
visitation at all times, it is concluded that night-flying insects were 
not a factor in the pollination of sweet clover at Arlington or at Ames 
in 1916. 

FLOWERS POLLINATED ONLY BY DAY-FLYING INSECTS. 

A cheesecloth-covered cage, 3 feet square and 3 J feet high, was 
placed on July 7, 1916, over two sweet-clover plants at Arlington, 
before any of the flowers opened. As the cover of this cage was 
removed at 7.30 a. m. and replaced at 4.30 p. m. each day during the 
experiment, the plants were subject to visitation by day-flying 
insects only. As soon as all of the flowers on most of the racemes had 
bloomed, and before any mature pods shattered, the racemes were 
removed from the plants and the pods produced by each raceme 
counted. The two plants produced a total of 544 racemes, with an 
average of 20.9 pods each. 

. 153321°— 20— Bull. 844 3 



18 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

This experiment was also conducted at Ames. One plant was 
protected from insect visitation at night in August, 1916, with the 
•result that it produced 418 racemes, with an average of 41.11 pods 
each. 

PLANTS PROTECTED FROM ALL INSECTS THAT COULD NOT PASS THROUGH A WIRE 
SCREEN HAVING 14 MESHES TO THE LINEAR INCH. 

It is well known that many small insects, and especially those 
belonging to the family Syrphidse and to the genus Halictus, frequent 
sweet-clover flowers, but no records have been noted that show how 
important these insects are as pollinators of this plant. In order to 
obtain data on this subject a cage 12 feet square and 6| feet high, 
made of wire screen having 14 meshes to the linear inch, was placed 
over a few plants at Ames, in July, 1916, before they began to bloom. 
The base of the cage was buried several inches in the soil, so that no 
insects could get into it. As these plants were growing in a field 
where there was a sufficient supply of moisture at all times, they made 
a growth of 5 to 6 feet. For this reason all the racemes were collected 
from only a portion of one of the plants instead of from the entire 
plant, as was done with the smaller ones inclosed in the cheesecloth- 
covered cages. The branches selected contained 224 racemes, with 
an average of 24.53 pods each. Many insects that were able to pass 
through the wire netting were observed working on the flowers of the 
inclosed plants. 

A check plant, subject to visitation by all insects and growing 
within a few yards of the cage, contained 264 racemes, with an average 
of 28.23 pods each. 

This experiment shows that small insects are efficient poUinators 
of sweet clover and that the plant to which all insects had access 
produced an average of only 3.7 pods to the raceme more than the 
one inclosed in the cage. As these plants were growing close to a 
strip of timber and some distance from a field of sweet clover, it is 
probable that more small insects worked on the flowers than would 
have been the case if the cage had been located in the center of a 
field of sweet clover. Though these results show that small insects 
are able to poUinate sweet-clover flowers freely, it is very doubtful 
whether insects of this kind would be numerous enough to pollinate 
sufficient flowers in a large field of sweet clover for profitable seed 
production. The honeybee is the most efficient pollinator of this 
plant, and it is believed that in" many sections it is responsible for the 
pollination of more than half of the flowers. 

SUMMARY OF INSECT-POLLINATION STUDIES. 

The data secured in the different experiments where sweet-clover 
flowers were subject to insect visitation at one time or another are 
presented in detail in Table V. 



SWEET-CLOVER SEED. 



19 



Table V. — Swrrmwry of the insect pollination studies conducted at Arlington, Va., and 

Ames, Iowa, in 191G. 



Location. 



Num- 
ber of 
plants. 



Method of treatment. 



Number of — 



Racemes. 



Pods 
pro- 
duced. 



Pods per 
raceme, 



Arlingon 

Ames 

Arlington 

Ames 

Arlington 

Do... 

Ames 

Arlin-' on 

A mes 

Do... 



Check — subject to insect visitation at 

all times. 

do 

Protected from all insects 

do 

Visited by night-flying insects only 

(cage 1). 
Visited by night-flying insects only 

(cage 2). 

Visited by night-flying insects only 

Visited by day-flying insects only 

do 

Protected from all insects , 



196 

239 

904 

2,370 

723 

227 

486 

544 

418 

1,594 



4,013 

9,943 
577 
653 

2,720 

152 

8,024 

11,397 

17, 186 

502 



20.47 

41.60 

.63 

.27 

3.76 

.07 

16.51 

20.95- 

41.11 

.31 



The results in Table V show that an average of 0.37 pod to the 
raceme was obtained from the plants protected from visitation by all 
insects during the flowering period. As the racemes of Melilotus 
alba will average approximately 50 flowers each, less than 1 per cent 
of them set seed without being pollinated by insects. The results 
obtained in the cages in which only night-flying insects had access to 
the flowers show that these insects pollinate sweet clover to a slight 
extent, but that the number of pods produced by them is so few that 
it may be assumed that these flowers would have been pollinated by 
day-flying insects. This assumption is borne out by the results 
obtained in the cages where only day-flying insects had access to the 
flowers, as the results obtained in these cages at Arlington and Ames, 
respectively, are approximately the same as those obtained on the 
plants subject to insect visitation at all times. It will be noted that 
the yield of seed on the plants visited by insects at Ames is much 
higher than that of the plants subjected to insect visits during the 
same period at Arlington. This difference in seed yield may be 
attributed to the fact that isolated plants were used in the experi- 
ments at Ames, and at Arlington the experiments were conducted 
with plants growing under field conditions. 

RELATION OF THE POSITION OF THE FLOWERS ON MELILOTUS ALBA 
PLANTS TO SEED PRODUCTION. 

Observations of sweet-clover plants grown under cultivation, and 
especially when the stands were thick, showed that the flowers of the 
racemes on the upper and exposed branches produced a larger per- 
centage of seed than those on the lower branches which were less 
exposed. It is thought by some that the failure of the flowers on the 
lower racemes to be fertilized is due to shading; but the results ob- 
tained in the cheesecloth and glass covered cages do not warrant this 



20 



BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 



belief, as it is doubtful whether the shading of the flowers on the 
lower racemes is more than that caused by the cheesecloth. It is 
probably the lack of pollination that causes this decrease in seed pro- 
duction, on the lower branches of plants growing close together, as a 
vast number of flowers open each day on portions of the plants which 
are exposed directly to visitation by insects and are therefore more 
accessible to them. 

In order to obtain information upon the number of flowers that 
produce seed on the upper and lower portions, respectively, of sweet- 
clover plants when grown under field conditions and where the stand 
contained four to five plants to the square foot, a number of racemes 
were labeled on different portions of the plants at Ames in 1915 and 
1916. Wlien the pods were partly mature, records were made of the 
number of flowers that produced pods. The results obtained are 
given in Table VI. 

Table VI. — Relation of the position of sioeet-clover flowers on the plants to seed pro- 
■ duction, at Ames, Iowa, in 1915 and 1916. 



Year. 



Position of the flowers. 



Nmnber 

of 
flowers. 



Pods formed. 



Number. 



Percent- 



Average. 



1915 
1916 



1915 
1916 



Upper half of plants 
do 

Lower half of plants 
do 



812 
261 



344 
216 



357 
101 



43.9 
38.7 



12.7 
27.3 



42.6 
18.3 



The flowers on the upper racemes of the plants produced 31.2 per 
cent more pods than those on the lower racemes in 1915, and 11.4 per 
cent more in 1916. These results prove that insects more frequently 
visit the flowers that are directly exposed and are therefore more 
accessible. 

INFLUENCE OF THE WEATHER AT BLOSSOMING TIME UPON SEED 

PRODUCTION. 

The seed production of sweet clover is seldom satisfactory when 
rainy or muggy weather prevails during the flowering period. In 
order to obtain data as to the relation existing between the visits of 
insects and the prevailing weather conditions, a record of insect visits 
and of the number of flowers that opened each day was kept for a 
period of nine days at Ames in August, 1915. 

In this experiment the racemes were marked early each morning 
just above the last flowers which had opened the previous day, and 
early the foUowing morning the number of flowers which had opened 
the previous day was noted. The number of flowers that were polli- 
nated was determined by the number of pods that formed. Table 
VII gives in detail the results obtained. 



SWEET-CLOVER SEED. 



21 



Table VII. — Inflixence of the weather at blossoming time upon the yield of sweet -clover 
seed, at Ames, Iowa, in 1915. 



Date, 
1915. 



Weather conditions. 



Insect visitors. 



Number 

of 
flowers 

that 
opened. 



Pods 
formed. 



Percent- 
age of 
flowers 
that 

matured. 



Aug. 16 
Aug. 17 
Aug. 18 
Aug. 19 
Aug. 20 
Aug. 21 
Aug. 22 
Aug. 2.3 
Aug. 24 



Cloudy and showery 

Rain all day 

Cloudy most of the day. . . 

Clear and cool 

Mostly clear and warm . . . 

Clear and warm 

Partly cloudy and warm . 

do 

Cloudy till mid-afternoon. 



Very few. . 

None 

Very few. . 
Numerous. 

....do 

....do 



■ do. 

Tew... 



102 
69 
60 
94 
61 
81 

181 

37 



18 
4 
20 
53 
38 
44 

100 

12 



17.6 
5.7 
33.3 
56.3 
62.2 
54.3 

55.2 

32.4 



The data given in Table VII show that the percentage of effective 
pollination is much higher in clear weather, when insects are active, 
than in cloudy or rainy weather, when but few insects visit the 
flowers. 

INSECT POLLINATORS OF SWEET CLOVER. 

On account of the ease with which the heavy flow of nectar of 
sweet-clover flowers may be obtained many insects visit the flowers, 
thereby pollinating them. While the useful insect visitors of flowers 
of red clover are limited to a few species of Hymenoptera, those 
pollinating sweet-clover blossoms are many and belong to such 
orders as Coleoptera, Lepidoptera, and Diptera, as well as to the 
Hymenoptera. However, in the United States the honeybee is the 
most important pollinator of sweet clover. In many parts of the 
country the different species of Halictus, commonly known as sweat 
bees, rank next in importance. The margined soldier beetles 
{Chauliognathus marginatus Fabr.) were very active pollinators at 
Arlington, Va., in the latter part of June and first part of July, 1916, 
but the woolly bear {Diacrisia virginica Fabr.) was the only night- 
flying insect found working on sweet clover at Arlington. 

Insects belonging to the genera Halictus, *Syritta, and Paragus 
were very active pollinators at Ames, Iowa, in 1916, and ranked 
next in importance to the honeybee.. In fact, the results obtained 
in the cage where the plants were protected from visitation by 
insects that could not pass through a screen having 14 meshes to 
the linear inch showed that these small insects were able under 
the conditions of that experiment to pollinate practically as many 
flowers as larger insects. 

The insects listed below were collected while visiting Melilotus 
alba and M. officinalis flowers in 1916. 



22 BULLETIN 844, U. S. DEPAKTMENT OF AGRICULTURE. 

AT ARLINGTON, VA. 

Neuroptera. — Perithemis domitia Dru., Enallagma sp. 

Hemiptera. — Adelphocoris rapidus Say, Lygus pratensis Linn, (tarnished plant 
bug). 

Coleoptera. — Chauliognathus marginatus Fabr. (margined soldier beetle), Diabrotica 
12-punctata Oliv. (southern com rootworm). 

Lepidoptera. — Pieris protodice Bd. (imported cabbage butterfly), Heodes hypophleas 
Bd., Lycaena comyntas Gdt., Hylephila campestris Bd., Scepsis fulvicollis Hubn., 
Ancyloxypha numitor Fabr., Pholisora catullxis Fabr., Ptjraustidsp., Loxostege similalis 
Gn. (garden webworm), Thecla melinus Hubn., Colias philodice Gdtl (the common 
sulphur butterfly), Tarachidia caudef actor Hubn., Pyraraeis atalanta Linn., Drasteria 
(2 species), Diacrisia virginica Fabr. (the woolly bear). 

Hymenoptera. — Halictus lerouxi Lep., H. provancheri (sweat bee), II. pectoralis 
Sm. (sweat bee), Halictus (3 unidentified species), H. legatus Say, Bombus affinis 
Cr., B. impatiens Harris (bumblebee), Melissodes bimaculata Lep., Polistes pallipes 
Lep. (paper wasp), Megachilesp. (leaf-cutter bee), Coelioxys octodentata Say, Xylocopa 
virginica Drmy (common carpenter bee), Pompiloides sp.. Apis mellifica Linn, (honey- 
bee), Philanthus punctatus Say, Sphex nigricans Dahlb. (caterpillar hawk), S. picti- 
pennis Walsh (caterpillar hawk). 

Diptera. — Archytas analis Fabr., Chrysomyia macellaria Fabr. (screw- worm fly), 
PoUenia rudis Fabr. (cluster fly), Ocyptera carolinae Desv., Trichophora ruficauda 
V. D. W., Eristalis arbustorum Linn., Physocephala tibialis Say. 

AT AMES, IOWA. 

Hemiptera. — Lygus pratensis Linn., Adelphocoris rapidus Say, 

Coleoptera. — Coccinella transversoguttata Fabr. 

Lepidoptera. — Eurymus eurytheme Bdv., Chrysophanus sp., Lycaena (2 species), 
Libythea bachmnni Kirtland, Pieris rapae Linn. 

Hymenoptera. — Angochlora sp.. Apis mellifica Linn., Colletes sp., Halictus lerouxi 
Lep., H. provancheri D. J., Halictus sp., Elis sp., Calliopsis andreniformis Smith, Polistes 
sp., Sphex sp., Eumenes fraterna Say, Sceliphron sp., Isodontia harrisi Fern., Cerceris 
sp., Oxybelus sp. 

Diptera. — Syritta sp., Paragus STp., Chrysomyia macellaria Desv., Syrphidse (2 uniden- 
tified specimens). 

EFFECT OF MOISTURE UPON THE PRODUCTION OF MELILOTUS ALBA 

SEED. 

Careful inspection of a number of sweet-clover fields in Iowa and 
Illinois in the autiunn of 1916 indicated that many plants were 
unable to obtain sufficient moisture for the proper development of 
their flowers. Examination of flowers that aborted shortly after 
reaching their mature size showed that the anther sacs had not 
burst, even though the pollen grains were mature. Apparently for 
the same reason many immature pods aborted. The precipitation 
for July, 1916, in Livingston County, 111., where the sweet-clover 
seed crop suffered materially for lack of moisture, was 3.2 inches less 
than normal, while the temperature was 4.5° F. above normal. In 
August the precipitation was 0.96 of an inch below normal and the 
temperatm-e 4.2° F. above normal. At Ames, Iowa, the precipita- 
tion was 3.54 inches below normal and the temperature 5.4° F. above 



SWEET-CLOVER SEED. 23 

normal in July. Both the precipitation and temperatui'e were about 
normal at Ames in August, but most of the precipitation fell before 
the experiments were conamenced. 

In north-central Illinois the seed production of sweet clover was 
very UTegular. Some fields produced an abundance of seed, while a 
large percentage of the pods on the plants in other fields near by, 
where the thickness of the stand, size of the plants, and conditions 
in general were approximately the same, aborted. It was evident 
that all stands producing a good seed crop were growing on weU- 
di'ained soil and that those which were not yielding satisfactorily 
were on poorly drained land. It is well known that sweet clover 
will produce deep taproots only when the plants are growing in 
well-drained soil and that a much-branched surface root system wiU 
be formed on poorly drained land, and especially when there is an 
excess of moistm'e or a high water table during the first season's 
growth. During this droughty period in 1916 the upper layer of soil 
became so depleted of moisture that the plants wdth surface root 
systems were unable to obtain sufficient water to mature their seed. 
On the other hand, the lack of precipitation and the high tempera- 
tures did not affect the moisture content of the subsoil sufficiently 
to interfere with the normal seed production of deep-rooted plants. 
According to Lutts (22, p. 47) this same condition was found to be 
true in Ohio in 1916. 

As a rule, under droughty conditions the second crop of sweet 
clover will produce a higher yield of seed than the first crop, as the 
second growth of the plants is seldom more than half as much as the 
first, thereby requiring less moisture. However, if showery hot 
weather prevails when the first crop is cut, the end of each stub is 
very apt to become infected, usually with a species of Fusarium, 
which kills all the cortex as far back as the upper bud or young shoot 
and that part of it on the opposite side of this bud to the bud below. 
If the second bud from the top of a stub is not directly opposite the 
upper one the decay may extend nearly to the ground. (PI. IV.) 
The destruction of half to two-thirds of the cortex from 2 to 4 inches 
below the upper bud materially reduces the quantity of water that 
can be conveyed to the branch above the base of the dead area. 
Plants thus infected obtain sufficient moisture for seed production 
only under the most favorable conditions. When the first crop is 
cut during warm dry weather, and especially when the first crop has 
not been permitted to make more than a 30 to 32 inch growth, the 
stubble seldom decays, and in no instance have the plants been 
observed to decay as far back as the upper buds. 

An experiment was conducted at Ames in the latter part of August 
and ffi"st part of September, 1916, to determine the effect of watering 
plants that were aborting a large percentage of their flowers and 



24 



BLT.LETIN 844, U, S. DEPARTMENT OF AGRICULTURE. 



immature pods. For this purpose several volunteer plants growing 
in a meadow were selected. A hole 12 inches square and 14 inches 
deep was dug 8 inches from the crown of one plant, and each evening 
during the experiment 2 gallons of water were poured into the hole. 
The top of the hole was kept covered, so as to check evaporation from 
it as, much as possible. Another plant of the same size and growing 
about 15 yards from the watered plant served as a check. On both 
plants many of the flowers and immature buds were aborting at the 
beginning of the experiment. The soil in this field was so depleted 
of moisture that the leaves of the plants wilted during the hottest 
part of the days preceding the experiment. The foliage on the check 
plant wilted each day for the first five days of the experiment. On the 
sixth day 0.96 of an inch of rain fell and four days later 0.23 of an 
inch more. The dropping of the flowers was temporarily checked by 
these precipitations, but owing to the dry, compact condition of the 
soil the rain was not sufficient to check entirely the fall of flowers and 
immature pods. At the beginning of the experiment the racemes on 
both plants were divided into three classes, according to the develop- 
ment of the flowers, and labeled. They were collected and the seeds 
counted as soon as the pods at the bases of the racemes commenced 
to turn brown. Table VIII presents the results obtained. 

Table VIII. — Effect of water upon the seed production of siveet clover when growing 
under droughty conditions at Ames, Iowa, in 1916. 



Stage of development when labeled. 



Flowers at the base of the racemes just ready to open . 

Pods 3 to 6 days old 

Pods 9 to 12 days old 



Plant not watered. 



Number 
of racemes 
labeled. 



Average 
number of 
pods per 

raceme 

that 
matured. 



27.39 
21.13 
15.23 



Plant watered. 



I Average 

Number '^^fj^^ 
ofracemes ^^1^^ 



labeled. 



110 
112 
50 



raceme 

that 
matured. 



55.63 
39.81 
29.86 



Increase 

from 
watering. 



28.24 
18.68 
14.63 



The effect of the water was noticeable soon after the first appli- 
cation, as the leaves and flowers on this plant became turgid and the 
anther sacs burst at the proper stage of their development. Very 
few flowers fell after the second day. The water decidedly checked 
the aborting of imnaature pods, as is shown by the results obtained 
on the racemes which were labeled after the pods had formed. The 
racemes which contained pods 3 to 6 days old when labeled matured 
9.95 pods to the raceme more than those which contained older pods 
at the beginning of the experiment, but this was expected, as most 
of the aborting which caused this difference had taken place before 
the racemes were labeled. As very few pods aborted before they 
were 3 to 6 days old, the difference of 9.95 pods to the raceme in favor 



SWEET-CLOVER SEED. 



25 



of the ones labeled when the flowers at their bases were just ready to 
open was largely due to the dropping of the flowers on the older 
racemes before the experiment was begun. 

It* will be seen that the production of mature pods on the plant 
not watered was much greater on the racemes that were labeled 
before the flowers opened than on the older racemes. This difference 
is undoubtedly due to the precipitation which fell on the sixth and 
tenth days of the experiment. It is believed that the yield of 15.23 
pods to the raceme on the ones labeled when the pods were 9 to 12 
days old is representative of the production of pods per raceme pre- 
vious to the precipitation and that the other racemes on this plant 
would have yielded proportionately if conditions had remained the 
same. 

In the early spring of 1916, Melilotus alba was planted in several 
large pots in the greenhouse of the Department of Agriculture at 
Washington, D. C. These pots were placed outside the greenhouse 
in the late spring, where they remained until the following January, 
when they were taken into the greenhouse. The plants grew rapidly 
and began to flower during the latter part of April, 1917. At this 
time two pots were placed in a large cage made of screen having 20 
meshes to the linear inch. One pot was submerged in a tub of water, 
so that the soil was saturated at all times, while the plant in the other 
pot was given only sufficient water to keep it from wilting. The 
pods on a few racemes were self -pollinated and the results obtained 
are given in Table IX. 



Table IX.- 



-Effect of moisture on the seed produxtion of Melilotus alba at Washington, 
D. C, in 1917. 



Soil treatment. 


Total number of — 


Flowers that ma- 
tured (per cent). 


Racemes. 


Flowers. 


Pods 
formed. 


Total. 


Increase. 




12 
17 


227 
425 


65 
234 


28.63 
55.05 




Soil saturated 


26.22 







The results of this experiment compare favorably with those ob- 
tained under field conditions at Ames in 1916. 



26 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

Part n.— STRUCTURE AND CHEMICAL NATURE OF THE SEED 
COAT AND ITS RELATION TO IMPERMEABLE SEEDS OF 
SWEET CLOVER.^ 

HISTORICAL SUMMARY. 

When agriculturists first began to cultivate wild legumes they 
observed that many seeds would not germinate within a compara- 
tively shdrt time after planting. Thus the problem of impermeable 
seeds began to demand attention many years ago. However, imper- 
meable seeds are not confined to the Leguminosse, as they occur also 
in the Malvaceae, Chenopodiacese, Convolvulacese, CannacesB, and 
other families. 

Since the first account of the structure of legume seed coats by 
Malpighi (23 v. 1) in 1687, many investigators have contributed to 
our knowledge of the structure of the coats of seeds belonging to this 
family. 

Pammel (31) made an extensive study of legume seeds, including 
all the genera in the sixth edition of Gray's Manual, as well as 
genera not included in that publication. He found that the seed coat 
uniformly consisted of three layers, namely, the outer layer of Mal- 
pighian cells, the osteosclerid layer, and the inner layer of nutrient 
cells. Pammel's work included a study of the seed coats of Meli- 
lotus alba and M. ojicinalis, and the descriptions and illustrations in 
his publication agree for the most part with the results obtained in 
the investigations reported in this article. However, more variation 
was noticed in the different layers of the seed coat than he describes. 

The cause of impermeability in seeds has been investigated by 
many. It has been found to be due to the embryos in some seeds, 
such as the hawthorns, but in most cases to the structui'e of the seed 
coat, and especially so in the Leguminosse. Crocker (3) states that, 
exactly opposite to the common view, the cause of delayed germina- 
tion generally lies in the seed coats rather than in the embryos. 
Nobbe (29) thought that the hardness of leguminous seeds was due 
to the Malpighian layer, and in a later publication Nobbe and Haen- 
lein (30, p. 81) state that the absorbent power of many seeds is inhib- 
ited or entirely arrested by the cones of the Malpighian cells and the 
shields built up between them, which consist principally of cutin. 
Huss (15) agrees with Nobbe and Haenlein. Verschaffelt (39) 
found that the impermeability of the seeds of Ca?salpiniacege and 
MimosacejB investigated was due to the inability of water to pass 
tlu-ough the canals of the seed coat. By soaking the seeds in alcohol 

or other substances which change the capillarity of the pores, the seed 

^ y . 

' The writers wish to acknowledge the service rendered by Mr. H. S. Doty, Instructor in Botany, Iowa 
State College, Ames, Iowa, in assisting in the preparation of this article. 



SWEET-CLOVER SEED. 27 

coats were made readily permeable to water. Gola (6) states that 
the cause of the impermeability of seeds is the peculiar character of 
the Malpighiaii cells, which prevents their infiltration and conse- 
quent increase in volume, while Bergtheil and Day (2) found that the 
hardness of the seeds of Indigofera arrecta was due to their possession 
of a very thin outer covering of a substance resistant to water. 
Ewart (5, p. 185) believes that in most impermeable seeds the cuticle 
prohibits the absorption of water, but gives as an exception Adan- 
sonia digitata, in which the whole integument seems to be permeable to 
water with difficulty. The following is quoted from White (42, p. 
205): 

As a general rule in small and medium-sized seeds the cuticle is well developed 
and represents the impermeable part of the seed coat, while in the cases of large seeds, 
such as those of Adansonia gregorii, Mucuna gigantea, Wistaria maideniana, and Guil- 
andina bonducella, the cuticle is relatively unimportant and inconspicuous. In these 
seeds the extreme resistance which they exhibit appears to be located in the palisade 
cells. 

In discussing the seed coat of Melilotus alba, Rees (33, p. 404) 
states that the outer layer consists of palisade cells covered exter- 
nally by a structureless membrane, which, however, did not appear 
to be cuticle but hemicellulose, as it stained magenta with chlor- 
iodid of zinc. The greater part of the walls of the palisade cells also 
appears to be composed of hemicellulose and the outer ends only 
were cuticularized. In order to find whether the outer membrane 
was in itself impermeable to water, this author treated seeds for short 
intervals in sulphuric acid to dissolve the outside covering without 
directly affecting the palisade cells. Seeds treated in this manner 
swelled in water and microscopic examination showed that the ends 
of the palisade cells were quite intact, but had separated from each 
other. From this it was concluded that the outer membrane is 
instrmnental in conferring impermeability on the seed, although not 
directly responsible for it, as is the case with a true cuticle. It is 
further believed that it probably served as a cement substance by 
means of which the cuticularized ends of the cells were held together 
closely, thus forming a barrier through which water could not pene- 
trate, but that as soon as this barrier was removed the ends of the 
palisade cells separated and water passed in between them. 

More than 20 years ago machines were devised by Kuntze, Michal- 
owski (27, p. 86), Huss (15), and later by Hughes (14), to scarify 
im.permeable seeds. Other methods have been recommended and 
employed to some extent for hastening the germination of seeds. 
HUtner (13, p. 44) treated seeds of red clover, white clover, and 
alfalfa 10, 30, and 60 minutes with concentrated sulphuric acid and 
found that the best germination resulted from the 60-minute treat- 
ment. Love and Leighty (21) also treated the seeds of various 



28 BULLETIN 844, V. S. DEPARTMENT OF AGRICULTURE. 

legumes with ooncentrated sulphuric acid and obtained a better 
germination in all cases. In their investigations with Melilotus alba 
it was found that a 2-hour treatment resulted in some injury to the 
seed, but that a treatment varying from 25 minutes to 1 hour gave 
good results. In most cases in our investigations the seed coats 
of sweet clover became permeable to water after a treatment of 
15 minutes in concentrated sulphuric acid, and within 5 minutes aU 
of the Malpighian cells were destroyed down to the light line. Har- 
rington (10) found that the soil, season, climate, color, or size of 
red-clover seeds had no influence upon the percentage of impermeable 
seeds and that the good germination ordinarily obtained with red 
clover was due to the scarifying of the seed coats by the rasps of 
hulling machines. Harrington (11) also studied the agricultural 
value of impermeable seeds and found that alternations of tempera- 
ture cause the softening and germinating of many impermeable 
clover seeds when a temperature of 10° C. or cooler is used in alter- 
nation with a temperature of 20° C. or warmer and that the effect 
of such an alternation of temperatm-e is greatly increased by pre- 
viously exposing the seeds to germinating conditions at a temperature 
of 10° C. or cooler and is decreased by previously exposing the seeds 
to germinating conditions at a temperature of 30° C. It is a weU- 
known fact that impermeable seeds which remain in the field over 
winter germinate readily the following spring. 

The light line is the most important and interesting feature of the 
Malpighian cell, at least so far as Melilotus alba and 31. officinalis are 
concerned. But one light line occurs in the Malpighian cells in 
most Leguminosse, although Pammel (32) reports two weU-developed 
light lines in Gymnocladus canadensis, Junowicz (16) found three in 
Lupinus varius, and Sempolowski (36) two in Lupinus angustifolius. 

Many investigators have studied the light line, and different 
theories have been advanced as to its function, physical properties, 
and chemical nature. Schleiden and Vogel (35, p. 26) in describing 
the mature testa of ScJiizolohium excelsum in 1838 undoubtedly referred 
to the light line when they stated that the walls of the Malpighian 
cells were not equally thickened. Mettenius (26), in 1846, was 
probably the first definitely to describe the light line. This author 
believed it was composed of pore canals, all appearing at the same 
height in the cells, but he was unable to prove this by cross sections. 
Lohde (20) studied the light line in seeds of Hibiscus trionum and 
found it cutinized. Hanstein (8) states that the Malpighian ceUs are 
composed of two cell layers and the light line is produced by the 
adjoining walls of the ends of the cells. Later, this same author (9), 
according to Harz (12), refers to the light line as a perforated disk 
composed of tissue of strong refracting power. 



SWEET-CLOVER SEED. 29 

Russow (34) concludes that the light line is produced by neither 
chemical nor mechanical changes but is caused by a modified molec- 
ular structure containing less water than the remainder of the'' cell 
wall. Hiltner (13) agrees with Russow's explanation. Harz (12, 
p. 561) also agrees with Russow and adds that he has observed that 
the light line disappeared in a number of cases after applications of 
nitric acid. Wigand and Dennert (43) suggested that the light line 
is due to a series of erect fissures, while Tietz (37, p. 32) believes it is 
due to a cheniical modification and that the phenomenon results 
from the exceptionally extreme density of parts of the cellulose 
membrane. Junowicz (16) found evidence of cellulose material. 
The cell wall at this point was strongly, refractive and had a different 
molecular structure. After studying Phaseolus vulgaris, Haberlandt 
(7, p. 38) agrees with the Russow explanation. In the seed of this 
plant the light line colored blue after being treated with chloriodid 
of zinc. Sempolowski (36) , who investigated the light line in Lupinus 
angustifolius , states that there is not only a difference in the molecular 
structure but also a chemical modification of the cell wall at this 
point, since with iodin and sulphuric acid the cell wall colored blue, 
whereas the light line colored yellow. Wettstein (41), who studied 
seeds of Nelumbo, agrees with Russow (34) and Sempolowski (36) 
that chemical and physical modifications occur. He found that iodin 
and sulphuric acid colored the Malpighian cells intensely blue, the 
light line at first yellowish, and then later it gradually became blue. 
This reaction may be accelerated by heat. Iodin produced the same 
ejffect, and the light line colored blue more rapidly. When treated 
with a water-withdrawing medium the light line was not altered for 
some time, but finally disappeared with continued application. 
Cooking for a long time in caustic potash or standing in cold caustic 
potash caused the cells to swell, while the light line remained unin- 
jured at first but finally disappeared. He also beheved that the 
absence of pore canals in. the region of the light line caused it to be 
more dense. 

Nobbe and Haenlein (30) treated sections of seed coats of Trifolium 
pratense with iodin and sulphuric acid and found that the light line 
colored blue as readily as the thickened ridges that radiate inward 
from it, but that the outer processes of the palisade cells projecting 
from the light line toward the cuticle stained dark brown. They also 
state that various causes work to produce such unusual lusters in 
the light line, the principle one of which is the thickened ridges which 
radiate inward, reach their greatest development at this point, and 
coalesce in the lumen of the cell. The result is that the light line 
falls upon a continuously homogeneous medium, while in the inner 
portions of the ridges the light passes through media of varying 
opacity, such as cellulose, water, and protoplasm, whereby it is pro- 



30 BULLETIN 844, U. S. DEPAKTMENT OF AGRICULTURE. 

gressively subdued in varying degrees by partial reflection. Pammel 
(31, p. 147) studied the light line in Melilotus alba and found that it 
consisted of a narrow but distinct refractive zone below the conical 
layer. The refractive zone colored blue with chloriodid of zinc. 
The whole upper part was, however, more or less refractive, while the 
remainder of the cell wall contained pigment and colored blue with 
chloriodid of zinc. Small canals project into the walls, in some 
cases extending beyond the light line. 

Beck (1) found that the light-refracting power of the light line was 
much greater than that of the undifferentiated membrane and stated 
that there may be in addition to this a cheniioal difference which can 
not be detected with the present microohemical methods. He does 
not beUeve that it is outioularized or that it contains less water than 
the rest of the cell. 

Marliere (24, p. 11) gives a physical explanation and states that the 
true cause of the light line lies in the peculiar structure of the sec- 
ondary membrane of the Malpighian cell. Tunmann (38, p. 559) 
observed that it did not hydrolize in weak acids and therefore decided 
that it was not hemicellulose. He found that it dissolved in concen- 
trated sulphuric acid more readily than the regions surrounding it 
and that it was composed of pectin or callose. In our investigations 
the main portion of the light Kne of Melilotus alba and M. officinalis 
was very resistant to concentrated sulphm-ic acid, only the narrow 
outer portion being attacked. It showed evidence of caUose. 

MATERIAL AND METHODS. 

Permeable and impermeable seeds ^ of Melilotus alba and M. of- 
fidTialis were obtained from commercial samples and also from sam- 
ples collected in the field. Those selected for sectioning were allowed 
to dry after being removed from the germinator and then embedded 
on the ends of pine blocks in glycerin gum, which was made by 
dissolving 10 grams of powdered gum arable in 10 c. c. of water and 
adding 40 drops of glycerin. After the glycerin gum had dried 
for 24 hours, the seeds were easily sectioned. This method of em- 
bedding causes no change in the seed coat. It is more satisfactory 
than the paraflan method for holding the seeds firmly. The glycerin 
gum dissolved readily when the sections were mounted in water. 

In the microchemical studies Sudan III, alcanin, chlorophyll solu- 
tion, and phosphoric acid iodin were used to test for cutin or suberin; 
sulphuric acid and iodin, chloriodid of zinc, and chloriodid of 
calcium for cellulose; phloroglucin and hydrochloric acid for lignin; 

I The term "permeable" is used in this paper to designate seeds whose coats are permeable to water in 
two weeks or less at temperatures favorable for germination, while the term "impermeable" is used to 
designate seeds whose seed coats are impermeable to water for this length of time when temperatures are 
favorable for germination. Impermeable seeds are commonly referred to as "hard seeds," and they may 
become permeable in time. 



SWEET-CLOVER SEED. 31 

ruthenium red for pectic substances; and sulphuric acid, Congo red, 
and aniline blue for callose. 

Wliere very thin sections were necessary for detailed study of the 
structure of the seed coat, pods in various stages of development 
were collected, and after the usual preliminary treatment they were 
embedded in paraffin and sectioned with the microtome. Micro- 
chemical tests were made with these sections by using various specific 
stains. Safranin was used to test for cutin, suberin, and lignin; 
haematoxylin and methyl blue for cellulose ; methylene blue, methyl 
violet B, mauvein, and ruthenium red for pectic substances; and 
aniline blue and Congo red for callose. In studying some points 
with reference to the pore system of the seed coat, it was necessary 
to use free-hand sections of fresh pods. 

In studying the seed coat in relation to the absorption of water, 
both permeable and impermeable seeds were soaked in water solu- 
tions of safranin, gentian violet, eosin, and haematoxylin, then dried 
and embedded in glycerin gum for sectioning. Seeds were soaked 
in stains dissolved in 95 per cent alcohol to test the penetration of 
alcohol. It was evident that the seed coats did not act as a filter, 
as the stains passed through them with the water or alcohol. 

STRUCTURE OF THE SEED COAT. 

There is very little endosperm present in mature seeds of Melilotus 
alba or M. officinalis. That which is present is quite permeable to 
water and therefore bears no relation to the impermeable seeds of 
these plants. 

The outer layer of the seed coat, which is the modified epidermal 
layer of the ovule, is known as the Malpighian layer. (PL V, figs. 1 
and 2.) The cells constituting this layer, commonly called palisade 
cells, are the most highly modified cells of the seed coat. They are 
very much elongated, their length varying in the different regions of 
the coat, and their outer tangential walls and the outer portions of 
their radial walls are so much thickened that their lumina are con- 
fiued to the iuner portion of the cells, sometimes occupying less than 
half the length of the cells. The inner tangential walls and inner 
portions of the radial walls are thickened just previous to the death 
of the cells, the thickening sometimes being only sHght and sometimes 
so much as to leave only very narrow lumina. 

There is a very thin layer on the outer surface of the Malpighian 
cells which has been called cuticle by previous investigators, but the 
chemical composition of this layer and its perviousness to water 
indicate that there is very little cutin present. Tliis layer is probably 
the primary epidermal cell wall rather than a deposit on the outer 
sm-face of the waU. To determine this a study of the development 
of the Malpighian cells is necessary. 



32 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

Beneath the so-called cuticle there is the much thickened outer 
portion of the Malpighian cells in which there are two rather distinct 
regions, one constituting the conelike structures and the other form- 
ing a continous layer over the conelike structures, separating them 
from the cuticle and filling in between them. These two regions 
separate easily, and in cutting sections the outer region, called by 
some the cuticularized portion, often breaks away, leaving the entire 
surface of the cones exposed. 

The term ''cuticularized layer" will be used to designate all of the 
thickening covering the cones, including that around the cones as 
well as the portion between the cones and the cuticle. This term is 
not entirely appropriate, for the region is practically free from cutin, 
but for the want of a better term it will be used. There are canals in 
the cuticularized layer and cones, which are easily seen when the 
sections are treated with chloriodid of zinc or sulphuric acid. A 
surface view of a section showing the cones and cuticularized layer 
when mounted in glycerin shows the canals as dark lines due to the 
air inclosed. The canals are most abundant along the lines where 
the lateral walls of the cells join, but many are within the cones and 
in the cuticularized substance between the cones. (PI. V, fig. 5.) 

The well-developed light line in Melilotus alba and M. officinalis is 
found just below the bases of the cones. In some seed coats only a 
few and in others none of the canals which are common in the cones 
and cuticularized region cross the light line. A very distinct line of 
small canals filled with air and thus forming a dark band is present 
just above the Ught line, thus making the light line more conspicuous. 
(PI. V, fig. 3.) When the lumina of the cells extend across the light 
line, they are exceedingly small. The light line is the most compact 
region of the Malpighian layer and is conspicuous because it refracts 
the light much more than the regions above and below it. 

Just below the Malpighian is a layer of cells variously modified 
and known as the osteosclerid. The cells of this layer are often 
referred to as the hourglass cells on account of their shape. In some 
regions of the seed coat they are expanded at both ends and their 
walls are much thickened, the thickenings forming ridges on the 
radial walls, while in other regions only the upper tangential wall and 
a portion of the radial walls are thickened and the cells are expanded 
only at the inner end, thus having the shape of the frustum of a cone. 
Beneath the osteosclerid layer is the nutrient layer. 

The nutrient layer contains chloroplasts. It varies not only in 
the number of layers of cells composing it, but also in the modifica- 
tions of these cells. This layer ranges from four to seven ceUs in 
thickness in the different parts of the seed coat. 



Bui . 844, U. S. Dept. of Agriculture 

.a \ 



Plate V. 




/o 



Structure of the Seed Coat of Sweet Clover, 

Fig. 1.— Section of the seed coat of Melilotus officinalis. X450. Fig. 2.— Another section of the seed 
coat of Melilotus officinalis, sliowLng the variation in size and modifications that occur in the tliree 
layers. X450. Fig. 3. — Section of the Malpigliian layer of a Melilotus alba seed, showing a line of 
canals just above the light zone. X450. Fig. 4.— Section of the Malpighian layer of a permeable 
Melilotus alba seed. X450. Fig. 5.— Tangential section of the Malpighian cells cut between the 
cuticle and tops of the cones, showing pores. X530. Fig. 6.— Section through the Malpighian layer 
of an impermeable Melilotus alba seed. X450. Fig. 7.— Section through the Malpighian layer of an 
impermeable Melilotus alba seed, showing the region through which water and stains readUy passed. 
X450. Fig. 8. — Cross section of a Malpighian cell of a permeable Melilotus alba seed through the 
region of the light zone , showing the lumen not entirely closed. X 530. Fig. 9.— Section through the 
Malpighian layer of a Melilotus alba seed shaded to show the portions which react to the cellulose and 
pectose tests. X450. Fig. 10.— Section through the Malpighian layer of a Melilotus alba seed which 
shows the condition of the seed coat after 60 minutes ' treatment of concentrated sulphuric acid. That 
portion above the light zone was destroyed, and the lumina as small pores through which much of 
the stain now passed were seen extending across the light line. The lines between the cells were 
much more distinct, appearing as small intercellular spaces through which some stain passed. X450. 
a. Cuticle; b, cuticularized layer; c, conelike portion of the thickening of the Malpighian cells; d, 
light line; e, region of a hard seed coat through which water and stains readily passed; I, lumen; M, 
Malpighian cells; jV, nutrient cells; O, osteosclerid cells; p, canals just above light zone. 



SWEET-CLOVER SEED. 33 

MICROCHEMISTRY OF THE SEED COAT. 

Tests for cutin showed that there was very little present in the 
seed coat. Slight reactions for cutin were observed in the cuticle, in 
the outer margin of the cuticularized layer, and in the basal portion 
of the cones. These reactions were so shght as to be almost negli- 
gible. It is evident that the cuticle and cuticularized layer are not 
well named in Melilotus alba and M. officinalis. Tests for cellulose 
showed that it was present in the cuticle, cuticularized layer, cones, 
the walls of the Malpighian cells bejow the light line, and the walls of 
the cells of the osteosclerid and nutrient layers. (PI. V, fig. 9.) 
The reaction for cellulose in the Malpighian ceUs was quite distinct in 
the walls below the light line, less distinct in the cones and cuticle, 
and least distinct in the cuticularized layer. 

Tests for lignin occasionally showed shght traces in the Malpighian 
cells below the light line. When treated with reagents for pectic sub- 
stances, the cuticle, cuticularized layer, cones, and all cell walls 
below the light hne gave a definite reaction. The reaction of the 
cones and cuticle was more pronounced than the cuticularized layer. 
Tests for callose gave no reaction except in the upper part of the 
light Hne. This part of the light line stained sHghtly blue with 
aniline blue and was easily dissolved with sulphuric acid. In cutting 
free-hand sections of fresh material the Malpighian layer sometimes 
broke along this line. The greater part of the Ught hne reacted to 
none of the tests, and its chemical nature was not determined. 

Wlien microtome sections of seeds in different stages of develop- 
ment were treated with various stains, the results were in accord 
with those obtained with free-hand sections. Thus with safranin 
the periphery and cones of the Malpighian cells were shghtly stained, 
while haematoxyhn and methyl blue stained aU the seed coat except 
the Hght line. The cones and cuticle stained more readily than the 
cuticularized layer, but neither stained as deeply as the ceU walls 
below the light line. Methylene blue, methyl violet B, and mauvein 
stained all above the hght hne, indicating the presence of pectic sub- 
stances; however, the staining was more prominent in the cones and 
cuticle. 

The difference in reaction of the cones and cuticularized layer to 
the cellulose and pectose tests probably indicates a difference in 
density rather than a difference in chemical composition. Since the 
cuticularized layer separates readily from the cones, there may be a 
difference in physical properties. 

With Congo red the upper part of the light hne was only very 
slightly stained, but aniline blue had a more pronounced effect. 

The microchemical tests apphed to the seed coat show that in the 
region above the light Hne there is only a sHght trace of cutin or 



34 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

suberin, but ti considerable amount of cellulose and pectose. All 
cell walls below the light line are mainly cellulose but contain some 
pectose. The upper portion of the light line contains callose, but 
the remainder of the Hght line appears to be chemically different 
from all other parts of the seed coat or else so dense as to resist the 
attack of the reagents. 

THE SEED COAT IN RELATION TO THE ABSORPTION OF WATER. 

A study of permeable seeds soaked in water containing stains 
showed that there were no local regions tlirough which the water 
passed. The stains passed through all regions of the seed coat. 
Coating the micropylar region with vasehne retarded germination, 
but had no effect upon the percentage of germination at the end of 
three days. In seed coats through which the stain had passed, the 
light line was not stained. Some stain was found in the canals which 
crossed the hght hne, and much more in the cell cavities. Th«re was 
no evidence that the stain had permeated the substance of the Ught 
hne. It was able to cross the light hne only when pores were present. 

In impermeable seeds the stains passed readily to the hght hne. 
(PI. V, fig. 7.) It was evident that the absorption of water was not 
prevented by either the cuticularized layer or the cone-shaped struc- 
tures of the Malpighian layer, but by the hght line. The region out- 
side of the light line became stained in a few hours, but there was no 
trace of the stain witliin the hght line after the seeds had remained 
a week in the stains. Alcohol did not penetrate the seed coat more 
readily than water. 

A COMPARISON OF PERMEABLE AND IMPERMEABLE SEED COATS. 

No difference in chemical structure was found between the coats of 
permeable and impermeable seeds. The principal differences were 
in the character and amount of thickening of the ceU walls. 

In many of the permeable seeds some of the canals were found to 
extend across the light line, but this was not true for all permeable 
seeds. (PI. V, fig. 8.) Oblique sections of permeable seed coats 
showed that the ceU cavities, although reduced to mere pores by the 
thickening of their radial walls, extended across the hght line into 
the base of the cones, thus forming a passageway through which the 
stains passed to the larger portions of the cell cavities below the light 
line. (PI. V, fig. 4.) 

In the coats of the impermeable seeds the hght hne was usually 
broader, the Malpighian cells thickened more below the light line, 
and the main cavities of the Malpighian ceUs were more reduced and 
farther below the hght hne than in the coats of permeable seeds. 
(PI. V, fig. 6.) No canals except occasionally a few very small ones 
were seen crossing the light line in impermeable seeds. Cross and 



SWEET-CLOVER SEED. 35 

oblique sections showed that the lumina of the Malpighian cell^ were 
closed in the region of the light line. Thus it was found that perme- 
able and impermeable seeds differ mainly in the amount of thickening 
which occurs in the walls of the Malpighian cells. In the imperme- 
able seeds the thickening which begins at the outer tangential wall 
of the Malpighian cell extends farther toward the inner tangential 
wall, leaving the cell lumina smaller and farther below the light line than 
in permeable seeds. The tliickening is also more complete in imper- 
meable seeds, leaving fewer and smaller canals across the light line 
as well as closing the cell lumina in the region of the light line. 

THE ACTION OF SULPHURIC ACID ON THE COATS OF IMPERMEABLE 

SEEDS. 

Impermeable seeds were soaked m concentrated sulphuric acid 
(sp. gr. 1.84) for 15, 30, and 60 mmutes; then washed and put in the 
staining solutions. After they had swollen, they were removed from 
the staining solutions, dried, and embedded m glycerin gum. A 
study of these seeds showed that the acid had eaten away all of the 
material outside of the light line and that the stain had passed 
through all regions of the seed coat. (PL V, fig. 10.) When observed 
under the microscope, it was seen that the action of the acid was 
rapid, destroymg the cuticle, cuticularized layer, and cones in about 
5 minutes. After 15 minutes treatment with acid the light line, 
aside from the presence of canals and pores not previously visible, 
seemed to be very little affected. The division lines along which the 
lateral walls of the Malpighian cells were jomed now became much 
more distinct across the light Ime, thus mdicating that there was 
some swellmg in this region. When a close examination of the path 
of the stain was made the cell lumma, and occasionally very small 
pores, were found to extend across the light Ime. The presence of 
the stam m the pores mdicated that they were paths of the stain 
across the light line. Some of the stam passed along the lines be- 
tween cells and through the occasional canals crossmg the light line, 
but judgmg from the mtensity of the stam in the lumina the canals 
appeared to be the prmcipal passageways. 

The action of the acid m opening the cell cavities across the light 
line was not deternmied. It may be due to the swellmg of the light 
line or to the removal of substances closing the pores. 

No seeds were exposed to the acid for longer than an hour, but at the 
end of this period the light line was still mtact. As compared with 
other portions of the Malpighian layer, it is extremely resistant to 
concentrated sulphuric acid. Smce all cell walls below the light Ime 
are mamly cellulose, the resistance of the light line prevents the acid 
from destroying the entire seed coat and reaching the embryo. 



LITERATURE CITED. 

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37 



38 BULLETIN 844, U. S. DEPARTMENT OF AGRICULTURE. 

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